In past years we have demonstrated the utility of genetic knockout animals combined with metabolic profiling for preclinical evaluation of potential new radiopharmaceuticals. Work is continuing on the study of the pharmacokinetics of our initial M2 selective ligand, FP-TZTP. Two collaborators are continuing human use protocols at the NIH Clinical Center. The uptake of FP-TZTP in monkey brain was found to be inhibited in a dose dependent manner by procaine. This result supports at least a partial muscarinic mechanism for the action of procaine. Our efforts in the development of muscarinic acetylcholine receptor ligands have turned to the search for M1 and M3 selective ligands. We evaluated two F-18 radiolabeled analogues of TZTP whose parent compounds were shown to be M1 functional agonists. The first, 7-fluoroheptylthioTZTP was found to lack receptor mediated uptake. We prepared an F-18 analogue of xanomeline, an M1 selective agonist that was clinically evaluated through phase III trials. Fluoroxanomeline demonstrated specific binding, but through use of knock-out mice proved to be M2 selective. We have also prepared our first M2 and M3 receptor antagonists. The M2 receptor antagonist demonstrates specific uptake in vivo. Unfortunately the uptake into the brain is not very high, suggesting that structural modifications will be required. FCWAY, our high affinity 5HT1A antagonist, has been clinically studied in an epilepsy protocol. The lower affinity FPWAY has been evaluated in mice for its uptake sensitivity to endogeneous ligand. Fluoxetine and paroxetine increase the serotonin in the synapse and thus should reduce the binding of FPWAY. In both rats and mice we were unable to detect uptake sensitivity to various doses of these two drugs. Our method of evaluating metabolism using hepatocytes and HPLC-MS-MS and HPLC with radioactivity detection have been applied to the evaluation of metabolic variations between species. We have compared the identity of metabolites and the relative rate of metabolism for a large number of compounds and using three species: rat, monkey, and human. In general, a particular compound gives the same metabolite profile regardless of the species. The exceptions are the WAY compounds, which undergo amide hydrolysis preferentially in human cells but aromatic oxidation predominates in monkeys and rats. Small Animal Imaging. In collaboration with the group of Michael Green, we have utilized the ATLAS small animal scanner to measure glucose metabolic rate in rat brain. We have also begun studies of the effects of anesthesia on the uptake of radiotracers. We have no technique to allow in vivo imaging of awake rats, so a comparison of scans post mortem of two groups of rats; one with an uptake period while awake and one with an uptake period while under isoflurane. We have collaborative projects that leverage the ability of our cyclotron facility to make a wide range of radionuclides. Both diagnostic and therapeutic nuclides were prepared for chemical and biochemical studies. In the diagnostic arena we prepared the first bromine-76 CRH analogue last year. In a continuation of this collaborative project, we have prepared other labeled analogues searching for one with better uptake and contrast properties that may lead to a useful imaging agent.